We are proposing to continue analysis of bacterial clones containing double-stranded cDNA copies of the mRNA products of a developmentally regulated, multigene family. This multigene family codes for approximately 100 distinct but evolutionarily related eggshell proteins in a silkmoth. These proteins are synthesized with distinct developmental kinetics. We will hybridize stage specific nuclear and cytoplasmic RNA isolated from eggshell-producing cells to cDNA clones in order to elucidate the temporal program of gene expression. This will be essential for determining at what level of information flow protein synthesis is regulated. We will also be using restriction enzyme mapping and DNA sequence analysis of these cDNA clones to determine pathways of evolutionary change for individual sequences within this multigene family. We will continue structural analysis of cloned chromosomal insertions containing chorion sequences, using the above-mentioned techniques as well as heteroduplex mapping. Major emphasis will be placed on determining the number of distinct chorion genes within a single chromosomal insertion as a direct test for physical linkage. In addition, we will identify particular chorion sequences found in the chromosomal insertions using our library of cDNA clones. We will also be constructing a library of cDNA clones in Bombyx mori, looking for differences in particular mRNA sequences or in sequence abundance between wild type and the chorion mutant GrB.